The alpha-2 subspecies of human leukocyte interferon (IFN-alpha-2) is a 165 amino acid polypeptide chain with a secondary structure characterized by two intramolecular disulfide bridges (CYS.sup.1 -CYS.sup.98 and CYS.sup.29 -CYS.sup.138) [G. Bodo and I. Fogy, "Characterization of Different Molecular Species in Affinity Purified Recombinant Human Interferon Alpha 2", The Interferon System, pp. 23-27 (1985); R. Wetzel, et al., "Properties of a Human Alpha-Interferon Purified From E.coli Extracts", J. Interfer. Res., 1, pp. 381-90 (1981)]. However, in the affinity purified polypeptide, Bodo and Fogy also found species of IFN-alpha-2 in which the disulfide bonds were mispaired, or in which the critical cystines were partially or completely reduced. Certain of these forms may have a lower specific activity [See H. Moorehead et al., "Roles of the 29-138 Disulfide Bond of Subtype A of Human .alpha. Interferon in Its Antiviral Activity and Conformational Stability", Biochemistry, 23, pp. 2500-07 (1984)]. Alternatively, they may present a conformation which is different from the native molecule, so that an adverse reaction could be elicited upon administration. Partially or completely reduced forms are less stable and can result in further scrambled molecules.
The literature distinguishes slow-moving monomers (SMM) having at least one free sulfhydryl group (partially or fully reduced form), and a fast-moving monomer (FMM) with two disulfide bonds (fully oxidized form). The free sulfhydryl groups of SMM could give rise to oligomers through the formation of intermolecular disulfide bonds. These oligomers have a lower specific activity, and may cause adverse reactions in humans [See S. Pestka and S. J. Tarnowski, "Purification Of The Interferons", Pharmac Ther., 29, pp. 299-319 (1986)]. Assays for oligomers of interferon are known [S. Pestka et al., "Specific Immunoassay for Protein Dimers, Trimers, and Higher Oligomers," Anal. Biochem., 132, pp. 328-33 (1983); S. Pestka, et al., "Procedures For Measurement Of Interferon Dimers And Higher Oligomers By Radioimmunoassay", Meth. Enzymol., 119, pp. 588-93 (1986)]. Considering all possible combinations of disulfide bonds and free sulfhydryl groups, theoretically ten monomeric forms of IFN-alpha-2 exist.
Upon extensive purification of the protein, D. R. Thatcher and N. Panayotatos isolated recombinant IFN-alpha-2, which they characterized as comprising a major component having a pI of 5.9 accompanied by three lesser anodic bands ["Purification of Recombinant Human IFN-.alpha.", Meth. Enzymology, 119, pp. 166-77 (1986)]. According to Thatcher and Panayotatos, the purification of correctly folded, fully oxidized monomer is not simple, because these "conformational variants" have properties which are very similar to those of the native molecule. Further, their work suggests that conformational variants may arise from the fact that the intracellular environment of host microorganisms has a net reducing redox potential, which favors the production of the reduced form of the monomer. Their observed conformational variants may arise from the reduced monomer.
In view of the clinical importance of pure fully oxidized form of IFN-alpha-2, attempts have been made to isolate FMM from other forms of the protein. Many procedures for the purification of IFN have been described in the literature. European patent application 108,585 refers to the removal of both oligomers and slow monomers from an interferon preparation by incubation at acid pH and elevated temperatures for a prolonged period. A. M. Felix et al., "Analysis Of Different Forms Of Recombinant Human Leukocyte Interferons by High Performance Liquid Chromatography", Methods in Enzymology, 119, pp. 242-48 (1986) reported the separation of SMM from FMM by a modification of the metal chelate chromatographic method of J. Porath et al. ["Metal Chelate Affinity Chromatography, A New Approach to Protein Fractionation," Nature (London), 258, pp. 598-99 (1975)]. U.S. Pat. No. 4,432,895 refers to the conversion of oligomeric interferon into monomeric interferon by treatment with a redox reagent. However, none has documented a method for separating fully oxidized fast moving monomers. Due to the difficulties in isolating homogeneous FMM in its native conformation in high quantities, the need for a method for its production still exists.